A potent trypanocidal component from the fungus Lentinus strigosus inhibits trypanothione reductase and modulates PBMC proliferation

The fungus Lentinus strigosus (Pegler 1983) (Polyporaceae, basidiomycete) was selected in a screen for inhibitory activity on Trypanosoma cruzi trypanothione reductase (TR). The crude extract of L. strigosus was able to completely inhibit TR at 20 µg/ml. Two triquinane sesquiterpenoids (dihydrohypnophilin and hypnophilin), in addition to two panepoxydol derivatives (neopanepoxydol and panepoxydone), were isolated using a bioassay-guided fractionation protocol. Hypnophilin and panepoxydone displayed IC50 values of 0.8 and 38.9 µM in the TR assay, respectively, while the other two compounds were inactive. The activity of hypnophilin was confirmed in a secondary assay with the intracellular amastigote forms of T. cruzi, in which it presented an IC50 value of 2.5 µ M. Quantitative flow cytometry experiments demonstrated that hypnophilin at 4 µM also reduced the proliferation of human peripheral blood monocluear cells (PBMC) stimulated with phytohemaglutinin, without any apparent interference on the viability of lymphocytes and monocytes. As the host immune response plays a pivotal role in the adverse events triggered by antigen release during treatment with trypanocidal drugs, the ability of hypnophilin to kill the intracellular forms of T. cruzi while modulating human PBMC proliferation suggests that this terpenoid may be a promising prototype for the development of new chemotherapeutical agents for Chagas disease.

A rapid detection of multidrug-resistant Mycobacterium tuberculosis by a nitrate reductase assay on blood agar

The susceptibility of 49 Mycobacterium tuberculosis clinical isolates to isoniazid (INH) and rifampisin (RIF) (28 multi-drug resistant-tuberculosis samples) was determined by a nitrate reductase assay (NRA) on blood agar. Agreement between the NRA and other testing methods was found to be 93.8% for both INH and RIF. The sensitivity, specificity, positive predictive value and negative predictive value for INH were 92.8%, 94.2%, 86.6% and 97%, respectively. The sensitivity, specificity, positive predictive value and negative predictive value for RIF were 90.4%, 96.4%, 95% and 93.1%. In conclusion, we show here that blood agar can be used effectively for the NRA test.

Change in mutation patterns of Plasmodium vivax dihydrofolate reductase (Pvdhfr) and dihydropteroate synthase (Pvdhps) in P. vivax isolates from malaria endemic areas of Thailand

Malaria is the most important public health problem in several countries. In Thailand, co-infections of Plasmodium vivax and Plasmodium falciparum are common. We examined the prevalence and patterns of mutations in P. vivax dihydrofolate reductase (Pvdhfr) and P. vivax dihydropteroate synthase (Pvdhps) in 103 blood samples collected from patients with P. vivax infection who had attended the malaria clinic in Mae Sot, Tak Province during 2009 and 2010. Using nested polymerase chain reaction-restriction fragment length polymorfism, we examined single nucleotide polymorphisms-haplotypes at amino acid positions 13, 33, 57, 58, 61, 117 and 173 of Pvdhfr and 383 and 553 of Pvdhps. All parasite isolates carried mutant Pvdhfr alleles, of which the most common alleles were triple mutants (99%). Eight different types of Pvdhfr and combination alleles were found, as follows: 57I/58R/117T, 57I/58R/117T, 57I/58R/117T/N, 57L/58R/117T, 57L/58R/117T, 58R/61M/117N, 58R/61M/117N and 13L/57L/58R/117T. The most common Pvdhfr alleles were 57I/58R/117T (77.7%), 57I/58R/117T/N (1%), 57L/58R/117T (5.8%) and 58R/61M/117N (14.5%). The most common Pvdhfr alleles were 57I/58R/117T (77.7%), 57I/58R/117T/N (1%), 57L/58R/117T (5.8%) and 58R/61M/117N (14.5%). Additionally, we recovered one isolate of a carrying a quadruple mutant allele, 13L/57L/58R/117T. The most prevalent Pvdhps allele was a single mutation in amino acid 383 (82.5%), followed by the wild-type A383/A553 (17.5%) allele. Results suggest that all P. vivax isolates in Thailand carry some combination of mutations in Pvdhfr and Pvdhps. Our findings demonstrate that development of new antifolate drugs effective against sulfadoxine-pyrimethamine-resistant P. vivax is required.

Comparative evaluation of the microplate nitrate reductase assay and the rezasurin microtitre assay for the rapid detection of multidrug resistant Mycobacterium tuberculosis clinical isolates

The microplate nitrate reductase assay (MNRA) and the rezasurin microtitre assay (REMA) were used for the susceptibility testing of 73 clinical isolates and the results were compared with those that were obtained using the Bactec 460 TB and Bactec MGIT 960 systems. The REMA and the MNRA were performed in 96-well plates. For the REMA, the concentrations of isoniazid (INH) and rifampicin (RIF) ranged from 1.0-0.01 µg/mL and 2.0-0.03 µg/mL, respectively. For the MNRA, the INH concentration was between 1.0-0.03 µg/mL and the RIF concentration was between 2.0-0.06 µg/mL. For the MNRA, the sensitivity, specificity, positive predictive value, negative predictive value and INH/RIF agreement were 100/95.6, 97.6/100, 96.8/100, 100/98 and 98.6/98.6, respectively, and for the REMA, they were 100/91.3, 90.4/100, 88.5/100, 100/96.1 and 94.5/97.2, respectively. Our data suggest that these two rapid, low-cost methods may be inexpensive, alternative assays for the rapid detection of multidrug resistant tuberculosis in low-income countries.

Biliverdin inhibits Toll-like receptor-4 (TLR4) expression through nitric oxide-dependent nuclear translocation of biliverdin reductase.

The cellular response to an inflammatory stressor requires a proinflammatory cellular activation followed by a controlled resolution of the response to restore homeostasis. We hypothesized that biliverdin reductase (BVR) by binding biliverdin (BV) quells the cellular response to endotoxin-induced inflammation through phosphorylation of endothelial nitric oxide synthase (eNOS). The generated NO, in turn, nitrosylates BVR, leading to nuclear translocation where BVR binds to the Toll-like receptor-4 (TLR4) promoter at the Ap-1 sites to block transcription. We show in macrophages that BV-induced eNOS phosphorylation (Ser-1177) and NO production are mediated in part by Ca(2+)/calmodulin-dependent kinase kinase. Furthermore, we show that BVR is S-nitrosylated on one of three cysteines and that this posttranslational modification is required for BVR-mediated signaling. BV-induced nuclear translocation of BVR and inhibition of TLR4 expression is lost in macrophages derived from Enos(-/-) mice. In vivo in mice, BV provides protection from acute liver damage and is dependent on the availability of NO. Collectively, we elucidate a mechanism for BVR in regulating the inflammatory response to endotoxin that requires eNOS-derived NO and TLR4 signaling in macrophages.

Peroxisome proliferator-activated receptor beta regulates acyl-CoA synthetase 2 in reaggregated rat brain cell cultures.

Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that regulate the expression of many genes involved in lipid metabolism. The biological roles of PPARalpha and PPARgamma are relatively well understood, but little is known about the function of PPARbeta. To address this question, and because PPARbeta is expressed to a high level in the developing brain, we used reaggregated brain cell cultures prepared from dissociated fetal rat telencephalon as experimental model. In these primary cultures, the fetal cells initially form random aggregates, which progressively acquire a tissue-specific pattern resembling that of the brain. PPARs are differentially expressed in these aggregates, with PPARbeta being the prevalent isotype. PPARalpha is present at a very low level, and PPARgamma is absent. Cell type-specific expression analyses revealed that PPARbeta is ubiquitous and most abundant in some neurons, whereas PPARalpha is predominantly astrocytic. We chose acyl-CoA synthetases (ACSs) 1, 2, and 3 as potential target genes of PPARbeta and first analyzed their temporal and cell type-specific pattern. This analysis indicated that ACS2 and PPARbeta mRNAs have overlapping expression patterns, thus designating the ACS2 gene as a putative target of PPARbeta. Using a selective PPARbeta activator, we found that the ACS2 gene is transcriptionally regulated by PPARbeta, demonstrating a role for PPARbeta in brain lipid metabolism.

Crystal packing modifies ligand binding affinity: the case of aldose reductase.

The relationship between the structures of protein-ligand complexes existing in the crystal and in solution, essential in the case of fragment-based screening by X-ray crystallography (FBS-X), has been often an object of controversy. To address this question, simultaneous co-crystallization and soaking of two inhibitors with different ratios, Fidarestat (FID; K(d) = 6.5 nM) and IDD594 (594; K(d) = 61 nM), which bind to h-aldose reductase (AR), have been performed. The subatomic resolution of the crystal structures allows the differentiation of both inhibitors, even when the structures are almost superposed. We have determined the occupation ratio in solution by mass spectrometry (MS) Occ(FID)/Occ(594) = 2.7 and by X-ray crystallography Occ(FID)/Occ(594) = 0.6. The occupancies in the crystal and in solution differ 4.6 times, implying that ligand binding potency is influenced by crystal contacts. A structural analysis shows that the Loop A (residues 122-130), which is exposed to the solvent, is flexible in solution, and is involved in packing contacts within the crystal. Furthermore, inhibitor 594 contacts the base of Loop A, stabilizing it, while inhibitor FID does not. This is shown by the difference in B-factors of the Loop A between the AR-594 and AR-FID complexes. A stable loop diminishes the entropic energy barrier to binding, favoring 594 versus FID. Therefore, the effect of the crystal environment should be taken into consideration in the X-ray diffraction analysis of ligand binding to proteins. This conclusion highlights the need for additional methodologies in the case of FBS-X to validate this powerful screening technique, which is widely used.

Crystal structure of yeast peroxisomal multifunctional enzyme: structural basis for substrate specificity of (3R)-hydroxyacyl-CoA dehydrogenase units.

(3R)-hydroxyacyl-CoA dehydrogenase is part of multifunctional enzyme type 2 (MFE-2) of peroxisomal fatty acid beta-oxidation. The MFE-2 protein from yeasts contains in the same polypeptide chain two dehydrogenases (A and B), which possess difference in substrate specificity. The crystal structure of Candida tropicalis (3R)-hydroxyacyl-CoA dehydrogenase AB heterodimer, consisting of dehydrogenase A and B, determined at the resolution of 2.2A, shows overall similarity with the prototypic counterpart from rat, but also important differences that explain the substrate specificity differences observed. Docking studies suggest that dehydrogenase A binds the hydrophobic fatty acyl chain of a medium-chain-length ((3R)-OH-C10) substrate as bent into the binding pocket, whereas the short-chain substrates are dislocated by two mechanisms: (i) a short-chain-length 3-hydroxyacyl group ((3R)-OH-C4) does not reach the hydrophobic contacts needed for anchoring the substrate into the active site; and (ii) Leu44 in the loop above the NAD(+) cofactor attracts short-chain-length substrates away from the active site. Dehydrogenase B, which can use a (3R)-OH-C4 substrate, has a more shallow binding pocket and the substrate is correctly placed for catalysis. Based on the current structure, and together with the structure of the 2-enoyl-CoA hydratase 2 unit of yeast MFE-2 it becomes obvious that in yeast and mammalian MFE-2s, despite basically identical functional domains, the assembly of these domains into a mature, dimeric multifunctional enzyme is very different.

Cloning and characterization of Helicobacter pylori succinyl CoA:acetoacetate CoA-transferase, a novel prokaryotic member of the CoA-transferase family.

Sequencing of a fragment of Helicobacter pylori genome led to the identification of two open reading frames showing striking homology with Coenzyme A (CoA) transferases, enzymes catalyzing the reversible transfer of CoA from one carboxylic acid to another. The genes were present in all H. pylori strains tested by polymerase chain reaction or slot blotting but not in Campylobacter jejuni. Genes for the putative A and B subunits of H. pylori CoA-transferase were introduced into the bacterial expression vector pKK223-3 and expressed in Escherichia coli JM105 cells. Amino acid sequence comparisons, combined with measurements of enzyme activities using different CoA donors and acceptors, identified the H. pylori CoA-transferase as a succinyl CoA:acetoacetate CoA-transferase. This activity was consistently observed in different H. pylori strains. Antibodies raised against either recombinant A or B subunits recognized two distinct subunits of Mr approximately 26,000 and 24, 000 that are both necessary for H. pylori CoA-transferase function. The lack of alpha-ketoglutarate dehydrogenase and of succinyl CoA synthetase activities indicates that the generation of succinyl CoA is not mediated by the tricarboxylic acid cycle in H. pylori. We postulate the existence of an alternative pathway where the CoA-transferase is essential for energy metabolism.

Nitrate reductase and glutamine synthetase activity in coffee leaves during fruit development

Nitrate reductase is the first enzyme in the pathway of nitrate reduction by plants, followed by glutamine synthetase, which incorporates ammonia to glutamine. The purpose of this study was to evaluate the nitrate reductase and glutamine synthetase activity, total soluble protein content, N and Ni content in coffee leaves during fruit development under field conditions to establish new informations to help assess the N nutritional status and fertilizer management. The experimental design was in randomized complete blocks, arranged in a 3 x 6 factorial design, with five replications. The treatments consisted of 3 N rates (0 - control, 150 and 300 kg ha-1) and six evaluation periods (January, February, March, April, May, and June) in six-year-old coffee (Coffea arabica L.) plants of Catuaí Vermelho IAC 44 cv. The nitrate reductase and glutamine synthetase activities, leaf soluble protein, and N concentrations increased linearly with the N rates. During fruit development, the enzyme activity, leaf soluble protein and N content decreased, due to the leaf senescence process caused by nutrient mobilization to other organs, e.g, to the berries. Leaf Ni increased during fruit development. Beans and raisin-fruits of plants well-supplied with N had higher Ni contents. Enzyme activities, total leaf N and leaf soluble protein, evaluated during the green fruit stage in March, were significantly correlated with coffee yield. These variables can therefore be useful for an early assessment of the coffee N nutritional status as well as coffee yield and N fertilization management.

Nitrate reductase activity and spad readings in leaf tissues of guinea grass submitted to nitrogen and potassium rates

Nitrogen and K deficiency are among the most yield limiting factors in Brazilian pastures. The lack of these nutrients can hamper the chlorophyll biosynthesis and N content in plant tissues. A greenhouse experiment was carried out to evaluate the relationship among N and K concentrations, the indirect determination of chlorophyll content (SPAD readings), nitrate reductase activity (RNO3-) in newly expanded leaf lamina (NL) and the dry matter yield for plant tops of Mombaça grass (Panicum maximum Jacq.). A fractionated 5² factorial design was used, with 13 combinations of N and K rates in the nutrient solution. The experimental units were arranged in a randomized block design, with four replications. Plants were harvested twice. The first harvest occurred 36 days after seedling transplanting and the second 29 days after the first. Significance occurred for the interaction between the N and K rates to SPAD readings and to RNO3- assessment taken on the NL during the first growth. Besides, RNO3- and SPAD readings increased only with the NL N concentration, reaching the highest values of both variables up to about 25 g kg-1, but were ratively constant at higher leaf N. Significant relationships either between SPAD readings or RNO3- activity and shoot dry mass weight were also observed. The critical levels of N concentration in the NL were, respectively, 22 and 17g kg-1 in the first and second harvest. Thus, SPAD instrument and RNO3- assessment can be used as complementary tools to evaluate the N status in forage grass.

Roles of multiple acyl-CoA oxidases in the routing of carbon flow towards β-oxidation and polyhydroxyalkanoate biosynthesis in Yarrowia lipolytica.

The oleaginous yeast Yarrowia lipolytica possesses six acyl-CoA oxidase (Aox) isoenzymes encoded by genes POX1-POX6. The respective roles of these multiple Aox isoenzymes were studied in recombinant Y. lipolytica strains that express heterologous polyhydroxyalkanoate (PHA) synthase (phaC) of Pseudomonas aeruginosa in varying POX genetic backgrounds, thus allowing assessment of the impact of specific Aox enzymes on the routing of carbon flow to β-oxidation or to PHA biosynthesis. Analysis of PHA production yields during growth on fatty acids with different chain lengths has revealed that the POX genotype significantly affects the PHA levels, but not the monomer composition of PHA. Aox3p function was found to be responsible for 90% and 75% of the total PHA produced from either C9:0 or C13:0 fatty acid, respectively, whereas Aox5p encodes the main Aox involved in the biosynthesis of 70% of PHA from C9:0 fatty acid. Other Aoxs, such as Aox1p, Aox2p, Aox4p and Aox6p, were not found to play a significant role in PHA biosynthesis, independent of the chain length of the fatty acid used. Finally, three known models of β-oxidation are discussed and it is shown that a 'leaky-hose pipe model' of the cycle can be applied to Y. lipolytica.

Evidence for catabolic pathway of propionate metabolism in CNS: expression pattern of methylmalonyl-CoA mutase and propionyl-CoA carboxylase alpha-subunit in developing and adult rat brain.

Methylmalonyl-CoA mutase (MCM) and propionyl-CoA carboxylase (PCC) are the key enzymes of the catabolic pathway of propionate metabolism and are mainly expressed in liver, kidney and heart. Deficiency of these enzymes leads to two classical organic acidurias: methylmalonic and propionic aciduria. Patients with these diseases suffer from a whole spectrum of neurological manifestations that are limiting their quality of life. Current treatment does not seem to effectively prevent neurological deterioration and pathophysiological mechanisms are poorly understood. In this article we show evidence for the expression of the catabolic pathway of propionate metabolism in the developing and adult rat CNS. Both, MCM and PCC enzymes are co-expressed in neurons and found in all regions of the CNS. Disease-specific metabolites such as methylmalonate, propionyl-CoA and 2-methylcitrate could thus be formed autonomously in the CNS and contribute to the pathophysiological mechanisms of neurotoxicity. In rat embryos (E15.5 and E18.5), MCM and PCC show a much higher expression level in the entire CNS than in the liver, suggesting a different, but important function of this pathway during brain development.

N-partitioning, nitrate reductase and glutamine synthetase activities in two contrasting varieties of maize

In order to identify useful parameters for maize genetic breeding programs aiming at a more efficient use of N, two maize varieties of contrasting N efficiency, Sol da Manhã NF (efficient) and Catetão (inefficient) were compared. Experiments were carried out under field and greenhouse conditions, at low and high N levels. The parameters analysed included total and relative plant and grain N content, biomass and the activities of nitrate reductase and glutamine synthetase in different parts of the plant. It was found that the translocation efficiency of N and photoassimilates to the developing seeds and the source-sink relations were significantly different for the two varieties. N content of the whole plant and grain, cob weight and the relative ear dry weight were useful parameters for characterizing the variety Sol da Manhã NF as to its efficient use of N. Enzymes activity of glutamine synthetase (transferase reaction) and nitrate reductase did not differ among the varieties.

Evolution of the ferric reductase domain (FRD) superfamily: modularity, functional diversification, and signature motifs.

A heme-containing transmembrane ferric reductase domain (FRD) is found in bacterial and eukaryotic protein families, including ferric reductases (FRE), and NADPH oxidases (NOX). The aim of this study was to understand the phylogeny of the FRD superfamily. Bacteria contain FRD proteins consisting only of the ferric reductase domain, such as YedZ and short bFRE proteins. Full length FRE and NOX enzymes are mostly found in eukaryotic cells and all possess a dehydrogenase domain, allowing them to catalyze electron transfer from cytosolic NADPH to extracellular metal ions (FRE) or oxygen (NOX). Metazoa possess YedZ-related STEAP proteins, possibly derived from bacteria through horizontal gene transfer. Phylogenetic analyses suggests that FRE enzymes appeared early in evolution, followed by a transition towards EF-hand containing NOX enzymes (NOX5- and DUOX-like). An ancestral gene of the NOX(1-4) family probably lost the EF-hands and new regulatory mechanisms of increasing complexity evolved in this clade. Two signature motifs were identified: NOX enzymes are distinguished from FRE enzymes through a four amino acid motif spanning from transmembrane domain 3 (TM3) to TM4, and YedZ/STEAP proteins are identified by the replacement of the first canonical heme-spanning histidine by a highly conserved arginine. The FRD superfamily most likely originated in bacteria.